JP2017111065A - Observation method of cuticle cell - Google Patents
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本発明は、角質細胞の観察方法に関する。更に詳細には、上皮再構築体を用いた、角質細胞の観察方法、角質細胞への被験物質の評価又はスクリーニング方法、被験物質の皮膚反応性の評価方法に関する。 The present invention relates to a method for observing corneocytes. More specifically, the present invention relates to a method for observing keratinocytes, a method for evaluating or screening a test substance on keratinocytes, and a method for evaluating skin reactivity of the test substance using an epithelial reconstructed body.
近年、各国における動物実験廃止の動き等もあり、動物を用いずに医薬品や化粧料の研究、評価等を行うための３次元培養皮膚モデルが開発されている（特許文献１）。この３次元培養皮膚モデルは、コラーゲンや繊維芽細胞を含む真皮同等物の層、表皮角化細胞の層等で構成されている。 In recent years, there has been a movement to abolish animal experiments in various countries, and a three-dimensional cultured skin model has been developed for research and evaluation of pharmaceuticals and cosmetics without using animals (Patent Document 1). This three-dimensional cultured skin model is composed of a layer of dermis equivalent containing collagen and fibroblasts, a layer of epidermal keratinocytes, and the like.
既に、このような３次元培養皮膚モデルに種々の剤を適用することや乾燥等の刺激を与えることによる、当該皮膚モデルの変化等についての研究が行われている。
例えば、前記３次元培養皮膚モデルに対し、紫外線を照射した後、メラニン量を確認することが行われている（特許文献１）。
Studies have already been conducted on changes in the skin model by applying various agents to such a three-dimensional cultured skin model and applying stimuli such as drying.
For example, the amount of melanin is confirmed after irradiating ultraviolet rays to the three-dimensional cultured skin model (Patent Document 1).
また、ヒト３次元培養表皮モデルに被験物質を接触させ、モデルの細胞中のガレクチン−７の減少量を指標として、皮膚バリア機能を改善又は経表皮水分蒸散量を抑制する剤をスクリーニングする方法が開発されている（特許文献２）。 In addition, there is a method in which a test substance is brought into contact with a human three-dimensional cultured epidermis model, and an agent for improving the skin barrier function or suppressing the transepidermal water transpiration amount is used with the decrease amount of galectin-7 in the model cell as an index It has been developed (Patent Document 2).
あるいは、ヒト培養皮膚モデルを乾燥させ、３次元トポグラフィー装置と原子間力顕微鏡を使い、直接的にヒト培養皮膚モデルの表面の形状と粗さを測定して形態的変化等を観察し、乾燥の有無による各種パラメーターを比較する検討等が行われている（非特許文献１）。 Alternatively, the human cultured skin model is dried, the shape and roughness of the surface of the human cultured skin model is directly measured using a three-dimensional topography apparatus and an atomic force microscope, and morphological changes are observed, followed by drying. The examination etc. which compare the various parameters by the presence or absence of are performed (nonpatent literature 1).
しかしながら、前記、メラニン量等を確認する検討方法（特許文献１）では、皮膚組織を破壊してメラニン量を測定することや、皮膚組織を固定し染色した観察用組織切片を作製することが行なわれたのみで、皮膚組織そのままを観察することは行われていなかった。 However, in the examination method for confirming the amount of melanin and the like (Patent Document 1), the skin tissue is destroyed and the amount of melanin is measured, or the skin tissue is fixed and stained for observation. However, it was not performed to observe the skin tissue as it was.
また、前記皮膚バリア機能を改善又は経表皮水分蒸散量を抑制する剤をスクリーニングする方法（特許文献２）では、３次元培養皮膚モデルの細胞中、もしくは培地中に分泌されるガレクチン−７や電気抵抗値を評価の対象としており、皮膚モデルの形態そのものを解析するものではなかった。 In addition, in the method for screening for an agent that improves the skin barrier function or suppresses the transepidermal water transpiration (Patent Document 2), galectin-7 or electricity secreted into the cells or medium of a three-dimensional cultured skin model The resistance value was the object of evaluation, and the form of the skin model itself was not analyzed.
更に、前記３次元トポグラフィー装置と原子間力顕微鏡を使ってヒト培養皮膚モデルを観察する研究（非特許文献１）では、乾燥処理した皮膚モデルを何ら表面処理等せずに顕微鏡で観察されたのみであった。 Furthermore, in a study of observing a human cultured skin model using the three-dimensional topography apparatus and an atomic force microscope (Non-Patent Document 1), the dried skin model was observed with a microscope without any surface treatment. It was only.
前記観察等の他に、前記３次元培養皮膚モデルは、従来、皮膚内を検討すること、例えば、細胞内分子に注目した評価等に、主に用いられていた。しかし、本発明者は、３次元培養皮膚モデルの表層を観察して評価すること案出した。そして、３次元培養皮膚モデルを用いて化粧料や皮膚外用剤等の評価やスクリーニングを行う際、より有用な角質細胞の観察方法を新たに見出し、本発明を完成した。 In addition to the above observations, the three-dimensional cultured skin model has been conventionally used mainly for examining the inside of the skin, for example, for evaluation focusing on intracellular molecules. However, the present inventor has devised to observe and evaluate the surface layer of the three-dimensional cultured skin model. And when evaluating and screening cosmetics, external preparations for skin, etc. using a three-dimensional cultured skin model, a more useful method for observing keratinocytes was newly found and the present invention was completed.
すなわち、本発明は、上皮再構築体の上面を除去し、上皮再構築体を顕微鏡で観察することを含む、角質細胞の観察方法を提供する。
また、本発明は、上皮再構築体に被験物質を適用した後、上皮再構築体の上面を除去して上皮再構築体を顕微鏡で観察することを含む、被験物質の評価又はスクリーニング方法を提供する。
That is, the present invention provides a method for observing keratinocytes, comprising removing the upper surface of the epithelial reconstructed body and observing the epithelial reconstructed body with a microscope.
The present invention also provides a test substance evaluation or screening method comprising applying a test substance to an epithelial reconstructed body, then removing the upper surface of the epithelial reconstructed body and observing the epithelial reconstructed body with a microscope To do.
本発明は、上皮再構築体に被験物質を適用した後、上皮再構築体の上面を除去して上皮再構築体を顕微鏡で観察することを含む、皮膚反応性の評価方法を提供する。
また、前記角質細胞の観察方法、前記被験物質の評価若しくはスクリーニング方法、又は前記皮膚反応性の評価方法に用いる試料を、上皮再構築体の上面を除去することによって作製することを含む、上皮再構築体試料の作製方法を提供する。
The present invention provides a method for evaluating skin reactivity, which comprises applying a test substance to an epithelial reconstructed body, then removing the upper surface of the epithelial reconstructed body and observing the epithelial reconstructed body with a microscope.
In addition, the sample used for the method for observing keratinocytes, the test substance evaluation or screening method, or the skin reactivity evaluation method is prepared by removing the upper surface of the epithelial reconstructed body. A method for producing a construct sample is provided.
本発明によれば、皮膚の外観を重視した角質細胞の観察を行うことができ、被験物質の評価やスクリーニング等の際に、客観的で正確なデータを得ることができる。
なお、ここに記載された効果は、必ずしも限定されるものではなく、本明細書中に記載されたいずれかの効果であってもよい。
According to the present invention, it is possible to observe stratum corneum with an emphasis on the appearance of the skin, and it is possible to obtain objective and accurate data during evaluation or screening of a test substance.
Note that the effects described here are not necessarily limited, and may be any of the effects described in the present specification.
本発明の角質細胞の観察方法は、上皮再構築体の上面を除去し、上皮再構築体を顕微鏡で観察することを含む。
上皮再構築体とは、いわゆる３次元培養皮膚モデルを含む概念である。本発明では、重層上皮再構築体を用いることが好ましいが、これに限定されない。
The method for observing corneocytes of the present invention includes removing the upper surface of the epithelial reconstructed body and observing the epithelial reconstructed body with a microscope.
The epithelial reconstructed body is a concept including a so-called three-dimensional cultured skin model. In the present invention, it is preferable to use a stratified epithelial reconstructed body, but the present invention is not limited to this.
＜上皮再構築体の作製方法＞
一例として、重層上皮再構築体（皮膚モデル）の作製方法を以下に示す。
(1) 上皮細胞、例えばケラチノサイト（表皮角化細胞）を初期増殖する。ここで、上皮細胞は哺乳動物由来が好ましく、ヒト由来が特に好ましい。
<Method for producing epithelial reconstructed body>
As an example, a method for producing a stratified epithelial reconstructed body (skin model) is shown below.
(1) Initial growth of epithelial cells such as keratinocytes (epidermal keratinocytes). Here, the epithelial cells are preferably derived from mammals, particularly preferably derived from humans.
(2) 前記(1)で増殖された上皮細胞を酵素処理にて剥がし、３次元培養用容器に播種し、培養する。
３次元培養用容器は、例えば、細胞培養用インサートを用いることができる。インサートの膜は、例えばポリカーボネート製のものが挙げられる。また、細胞培養用インサートではなく、コラーゲンゲル上又は生体より摘出した真皮にガラス製リングを載せ、その中に上皮細胞を播種してもよい。
(2) The epithelial cells grown in (1) above are peeled off by enzyme treatment, seeded in a three-dimensional culture vessel, and cultured.
As the three-dimensional culture container, for example, a cell culture insert can be used. Examples of the membrane of the insert include those made of polycarbonate. Further, instead of the cell culture insert, a glass ring may be placed on the dermis extracted from the collagen gel or from the living body, and epithelial cells may be seeded therein.
(3) 前記培養容器内で上皮細胞が隙間なく増殖したら、培地を分化用培地（高Ｃａイオン含有、例えばＣａイオン濃度約1.2 mM 〜約1.8 mM）に代え、例えば１６〜２０時間培養する。培養中、細胞は培地に浸漬している状態である。
なお、当該(3)の工程は、必須ではなく、前記工程(2)から後述する工程(4)に移ることも可能だが、工程(3)を経ることが好ましい。
(3) When epithelial cells grow without gaps in the culture vessel, the medium is replaced with a differentiation medium (high Ca ion content, for example, Ca ion concentration of about 1.2 mM to about 1.8 mM), and cultured for 16 to 20 hours, for example. During the culture, the cells are immersed in the medium.
Note that the step (3) is not essential, and it is possible to move from the step (2) to the step (4) described later, but it is preferable to go through the step (3).
(4) 前記工程(2)又は(3)の細胞培養インサート内の培地を取り除き、培養上皮細胞の上面を空気に曝露させる。このとき、細胞の底面のみが分化用培地に触れている状態となる。
空気曝露後、１２〜１４日後程度で、ヒトと同様の構造の重層上皮再構築体が得られる。
以上の重層上皮再構築体の作製方法の概略を図１に示す。
(4) The medium in the cell culture insert in the step (2) or (3) is removed, and the upper surface of the cultured epithelial cells is exposed to air. At this time, only the bottom surface of the cell is in contact with the differentiation medium.
After about 14 to 14 days after exposure to air, a stratified epithelial reconstructed body having a structure similar to that of a human is obtained.
FIG. 1 shows an outline of the method for producing the stratified epithelium reconstructed body.
このような重層上皮再構築体は、よりヒトの生体の皮膚に近い状態のものであり、化粧料、皮膚外用剤等の評価やスクリーニング等に適する。
なお、前記作製方法は、特開２０１１−１２０５７７号明細書を参考にすることができる。
Such a stratified epithelial reconstructed body is in a state closer to human skin, and is suitable for evaluation and screening of cosmetics, external preparations for skin, and the like.
In addition, the said manufacturing method can refer to Unexamined-Japanese-Patent No. 2011-120777.
＜上皮再構築体の観察前の処理＞
重層上皮再構築体を顕微鏡で観察する前に、重層上皮再構築体の上面を除去する。重層上皮再構築体の最上層には、壊死した細胞や、空気曝露による急激な環境変化により不完全に角化したと思われる細胞の堆積物が見られる。これを、物理的に一部又は全部除去することにより、角質細胞を観察しやすくすることができる。
従来の重層上皮再構築体の目視観察又は顕微鏡観察では、重層上皮再構築体の上面は一見なだらかに見えていたため、そのまま上面を観察することしか行われておらず、重層上皮再構築体の上面を除去するという発想がなかった。
<Treatment before observation of epithelial reconstructed body>
Prior to observing the stratified epithelial reconstruction with a microscope, the upper surface of the stratified epithelial reconstruction is removed. In the uppermost layer of the stratified epithelium reconstructed body, necrotic cells and deposits of cells that appear to be incompletely keratinized due to a rapid environmental change due to air exposure are seen. By physically removing part or all of this, the corneocytes can be easily observed.
In the conventional visual observation or microscopic observation of the stratified epithelial reconstructed body, since the upper surface of the stratified epithelial reconstructed body was apparently visible, only the upper surface was observed as it was, and the upper surface of the stratified epithelial reconstructed body was observed. There was no idea to remove.
重層上皮再構築体の上面の除去方法は特に限定されないが、好ましくは、スライドグラスや粘着剤の付いたテープ等を重層上皮再構築体の上面に一定圧で軽く押し当てて、上面を除去する方法を用いる。一般的には、テープストリッピングが簡易で好ましく用いられる。
スライドグラスやテープを重層上皮再構築体の上面に押し当てるときの圧力を調節することにより、除去する面積を増減できる。
ここで、重層上皮再構築体の上面を除去する厚さ（深さ）は、上面から１μｍ以上が好ましく、角質細胞の観察に適したものとなる。
The method for removing the upper surface of the stratified epithelial reconstructed body is not particularly limited, but preferably, the upper surface is removed by lightly pressing a slide glass or a tape with an adhesive on the upper surface of the stratified epithelial reconstructed body at a constant pressure. Use the method. Generally, tape stripping is simple and preferably used.
The area to be removed can be increased or decreased by adjusting the pressure when pressing the slide glass or tape against the upper surface of the stratified epithelial reconstructed body.
Here, the thickness (depth) for removing the upper surface of the stratified epithelial reconstructed body is preferably 1 μm or more from the upper surface, and is suitable for observation of keratinocytes.
また、より下層の角質細胞を観察したいときは、上面の除去を１回だけでなく、数回行うとよい。
特に、テープストリッピングにおいては、１回の操作でテープの粘着性により剥離される角質細胞が、通常１層となるので、テープストリッピングの回数により除去できる角質細胞の深さを決めることができる。すなわち、この操作を複数回繰り返すことにより、角質を上層から内部（深さ）方向にかけて段階的に角質細胞を除去することができる。
Moreover, when it is desired to observe the lower layer of keratinocytes, the upper surface is removed not only once but several times.
In particular, in tape stripping, the horny cells that are peeled off due to the adhesiveness of the tape in a single operation usually form a single layer, and therefore the depth of horny cells that can be removed can be determined by the number of times of tape stripping. That is, by repeating this operation a plurality of times, the horny cells can be removed stepwise from the upper layer to the inner (depth) direction.
重層上皮再構築体の上面を除去した後、当該重層上皮再構築体は顕微鏡観察に供される。
今まで一般には、テープストリッピング等をした後は、テープの方を顕微鏡観察等に供し、テープに付着した角化細胞等を見ることが行われていた。
しかし、本発明では、テープではなく、重層上皮再構築体の方を顕微鏡観察に供することが特徴的である。
After removing the upper surface of the stratified epithelial reconstructed body, the stratified epithelial reconstructed body is subjected to microscopic observation.
In general, after tape stripping or the like, the tape has been subjected to microscopic observation or the like to see keratinocytes or the like attached to the tape.
However, the present invention is characterized in that not the tape but the stratified epithelial reconstructed body is subjected to microscopic observation.
上皮再構築体の観察は、詳細は後述するが、共焦点レーザー顕微鏡を用いることが好ましく、特に反射式共焦点レーザー顕微鏡が好ましい。共焦点レーザー顕微鏡を用いることにより、上皮再構築体の個々の角質細胞の形等を詳細に観察することができる。
前記テープストリッピングは、共焦点レーザー顕微鏡で重層上皮再構築体の角質細胞を観察する場合、少なくとも１回、好ましくは２回、３回程度行えばよい。
Although details of the observation of the epithelial reconstructed body will be described later, it is preferable to use a confocal laser microscope, and in particular, a reflective confocal laser microscope is preferable. By using a confocal laser microscope, the shape of individual keratinocytes of the epithelial reconstructed body can be observed in detail.
The tape stripping may be performed at least once, preferably twice or three times when observing keratinocytes of the stratified epithelial reconstructed body with a confocal laser microscope.
本発明の観察方法を実際に適用するときは、上皮再構築体の観察前に、例えば、化粧料や皮膚外用剤の成分等の被験物質を上皮再構築体の上面に数分間〜数週間適用する。
具体的には、例えば、被験物質溶液を培養細胞インサート内に２００μＬ程度入れることや、被験物質を直接塗布等して、一定時間静置する。被験物質溶液の場合は、静置後、溶液を取り除く。この操作を１日数回行う。
When actually applying the observation method of the present invention, before observing the epithelial reconstructed body, for example, a test substance such as a cosmetic or a topical skin preparation is applied to the upper surface of the epithelial reconstructed body for several minutes to several weeks. To do.
Specifically, for example, about 200 μL of the test substance solution is placed in the cultured cell insert, or the test substance is directly applied, and allowed to stand for a certain time. In the case of a test substance solution, remove the solution after standing. This operation is performed several times a day.
次に、被験物質適用後の上皮再構築体を細胞培養インサートから支持膜ごと切り出す。
上皮再構築体の上面をテープストリッピング等で除去し、上皮再構築体を顕微鏡観察に供する。被験物質適用時、静置中、適用後、観察に供するまでは、例えば相対湿度を１０％以上９０％以下、３０％以上７０％以下、４０％以上５０％以下に調節してもよい。
なお、前記被験物質を上皮再構築体に適用して顕微鏡観察に供するまでの概略を図２に示す。
Next, the epithelial reconstructed body after application of the test substance is cut out together with the supporting membrane from the cell culture insert.
The upper surface of the epithelial reconstructed body is removed by tape stripping or the like, and the epithelial reconstructed body is subjected to microscopic observation. For example, the relative humidity may be adjusted to 10% or more and 90% or less, 30% or more and 70% or less, or 40% or more and 50% or less before application to the test substance, during standing, after application, or before observation.
In addition, FIG. 2 shows an outline from applying the test substance to the epithelial reconstructed body to use in the microscopic observation.
あるいは、上皮再構築体の上面（表面）への適用以外による被験物質の投与の検討にも前記方法を用いることができる。
例えば、美肌効果が見込まれる被験物質を、経口投与、静脈内投与、筋肉内投与、皮下投与、髄腔内投与等と同じような適用方法になるように投与を行ってから、上皮再構築体の上面を除去し、顕微鏡観察に供してもよい。すなわち、生体の皮膚の内側から被験物質が作用する様子を再現するモデルとして、上皮再構築体の下側から被験物質が取り込まれるように適用することもできる。
Alternatively, the above-described method can be used for examination of administration of a test substance other than application to the upper surface (surface) of the epithelial reconstructed body.
For example, a test substance that is expected to have a beautifying effect is administered in a manner similar to oral administration, intravenous administration, intramuscular administration, subcutaneous administration, intrathecal administration, etc. The upper surface may be removed and used for microscopic observation. That is, it can be applied so that the test substance is taken in from the lower side of the epithelial reconstructed body as a model for reproducing the action of the test substance from the inside of the skin of the living body.
本発明は、被験物質の上皮再構築体への適用の前後に上皮再構築体の角質細胞を観察・比較することにより、該被験物質による皮膚への影響を確認したり、作用を研究したり、美肌効果の優劣等を評価することに用いることができる。
また、本発明は、種々の被験物質を上皮再構築体に適用して観察することにより、所望の影響を皮膚に与える物質をスクリーニングすることに用いることもできる。
更に、本発明の観察方法は、特定の被験物質を、様々な種類の上皮再構築体、例えば異なる複数の哺乳動物由来の上皮再構築体に適用し、上皮再構築体の皮膚反応性の評価をすることに用いることもできる。
The present invention confirms the effect of the test substance on the skin and studies the action by observing and comparing the keratinocytes of the epithelial reconstructed body before and after application of the test substance to the epithelial reconstructed body. It can be used to evaluate the superiority or inferiority of the skin beautifying effect.
In addition, the present invention can also be used for screening substances having a desired effect on the skin by applying various test substances to the epithelial reconstructed body and observing them.
Furthermore, the observation method of the present invention applies a specific test substance to various types of epithelial reconstructed bodies, for example, epithelial reconstructed bodies derived from different mammals, and evaluates the skin reactivity of the epithelial reconstructed bodies. It can also be used to
＜顕微鏡による角質細胞の観察＞
上皮再構築体の角質細胞を肉眼で観察することは困難であるので、顕微鏡を用いることが好ましい。顕微鏡は特に限定されないが、前述のように、共焦点レーザー顕微鏡、特に反射式共焦点レーザーが好ましい。共焦点レーザー顕微鏡を用いることにより、上皮再構築体の角質細胞の詳細な形状を固定などの特別な処理をすることなく、大気圧中で、短時間に観察することができる。
<Observation of corneocytes by microscope>
Since it is difficult to observe the keratinocytes of the epithelial reconstructed body with the naked eye, it is preferable to use a microscope. Although the microscope is not particularly limited, as described above, a confocal laser microscope, particularly a reflective confocal laser is preferable. By using a confocal laser microscope, the detailed shape of the keratinocytes of the reconstructed epithelium can be observed in a short time at atmospheric pressure without any special treatment such as fixation.
角質細胞の観察要素は、例えば、角質細胞の形、配向及び面積、個々の角質細胞の面積のばらつき、並びに観察視野の単位面積当たりの細胞数等が挙げられる。
バリア能が健全な皮膚では、角質細胞の形は六角形に近いので、重層上皮再構築体の角質細胞が六角形に近いほど好ましく、例えば肌の見た目の美しさにも関係する。
また、角質細胞の配向は、整然とした配列を成していることが好ましい。
角質細胞の面積は、大きいほど好ましく、より成熟度の高い角質細胞であるといえる。
個々の角質細胞の面積のばらつきは、少ないほど好ましく、肌表面形状の均一性に関係していることが考えられる。
観察視野の単位面積当たりの細胞数は、被験物質の適用の前後で減少していることが好ましい。
The corneocyte observation elements include, for example, the shape, orientation and area of the corneocytes, the variation in the area of individual corneocytes, and the number of cells per unit area of the observation field.
In skin with a good barrier ability, the shape of the keratinocytes is close to a hexagon, so that the horny cells of the stratified epithelial reconstructed body are closer to the hexagon, which is also related to, for example, the appearance of the skin.
In addition, the orientation of the corneocytes is preferably an orderly arrangement.
The area of the keratinocytes is preferably as large as possible, and can be said to be a keratinocyte having a higher maturity.
The variation in the area of individual keratinocytes is preferably as small as possible, and may be related to the uniformity of the skin surface shape.
The number of cells per unit area of the observation field is preferably decreased before and after application of the test substance.
本発明の角質細胞の観察方法を適用すれば、上述の他にも、蛍光で標識された薬剤（被験物質）を上皮再構築体に塗布し、角質層内に浸透させ、薬剤の角質層での貯留状態と角質細胞形態変化とを併せて評価することが可能である。また、上皮再構築体に被験物質を適用後、各種タンパク質を蛍光免疫染色すれば、上皮再構築体内のタンパク質発現の変化と角質細胞形態変化とを併せて評価することも可能である。 If the keratinocyte observation method of the present invention is applied, in addition to the above, a fluorescently labeled drug (test substance) is applied to the epithelial reconstructed body and penetrated into the stratum corneum. It is possible to evaluate both the storage state and the keratinocyte morphology change. In addition, if various proteins are fluorescently immunostained after applying the test substance to the epithelial reconstructed body, it is possible to evaluate both the protein expression change and the keratinocyte morphology change in the epithelial reconstructed body.
＜角質細胞観察装置＞
本発明の適用の一態様として、角質細胞観察装置を提供することもできる。
角質細胞観察装置は、例えば、上皮再構築体の上面が除去された該上皮再構築体を入れる容器部と、該上皮再構築体の角層細胞を観察する顕微鏡部と、該顕微鏡部により取得された画像から、角質細胞の形、配向、面積、面積のばらつき及び観察視野の単位面積あたりの細胞数（細胞密度）からなる群から選択される観察要素を少なくとも１つ解析する画像解析部とを含む。
容器部は特に限定されないが、上皮再構築体の細胞がインタクトに保たれる観察用セルが好ましい。
顕微鏡部は、特に限定されないが、前述のように共焦点レーザー顕微鏡が特に好ましい。
画像解析部には、例えば、顕微鏡部により得られる像を撮像する画像取得部、画像記録部、画素の配列や画素数を分析する画素分析部、前記観察要素を画素数から数値化する計算部、観察視野の単位面積当たりの細胞を計数する細胞計数部等を含めることができる。
<Keratinocyte observation device>
As one aspect of application of the present invention, a keratinocyte observation device can also be provided.
The keratinocyte observation apparatus is obtained by, for example, a container part for storing the epithelial reconstructed body from which the upper surface of the epithelial reconstructed body is removed, a microscope part for observing the horny layer cells of the epithelial reconstructed body, and the microscope part. An image analysis unit that analyzes at least one observation element selected from the group consisting of the shape, orientation, area, variation in area, and number of cells per unit area of the observation field (cell density) from the obtained images including.
The container part is not particularly limited, but an observation cell in which cells of the epithelial reconstructed body are maintained intact is preferable.
The microscope section is not particularly limited, but a confocal laser microscope is particularly preferable as described above.
The image analysis unit includes, for example, an image acquisition unit that captures an image obtained by the microscope unit, an image recording unit, a pixel analysis unit that analyzes the pixel arrangement and the number of pixels, and a calculation unit that digitizes the observation element from the number of pixels A cell counting unit that counts cells per unit area of the observation field can be included.
細胞の径や周は、例えば、画像中の距離（単位距離）や画素間隔に基づいて算出可能である。
細胞の形は、例えば、特定の画像領域を構成する画素の配列に基づいて特定することが可能である。また、画像領域上の各位置における微分係数を演算して細胞の形を算出することもでき、パターンマッチング処理などを行ってもよい。細胞が六角形に近いかどうかを判断するには、例えば、六角形状のテンプレート画像との画像相関処理を行うことにより可能である。
細胞の配向は、例えば、画像領域を細線化してワイヤモデルを作成し、このワイヤモデルに基づいて求めることができる。
細胞の面積は、例えば、単位面積に含まれる画素数をカウントして単位面積画素数を取得しておくとともに、細胞領域内の画素数をカウントし、この画素数を単位面積画素数で除算することにより算出でき、通常の積分演算を行って面積を求めることも可能である。
細胞の面積のばらつきは、例えば、複数の細胞の面積を統計処理して算出することが可能である。
細胞密度は、観察視野の単位面積を特定し、その面積中の細胞数から算出すればよい。
これらの算出に用いられるソフトウェアは既知であり、それを適用すればよい。
The diameter and circumference of the cell can be calculated based on, for example, a distance (unit distance) or a pixel interval in the image.
The shape of the cell can be specified based on, for example, the arrangement of pixels constituting a specific image region. In addition, the shape of the cell can be calculated by calculating a differential coefficient at each position on the image region, and pattern matching processing or the like may be performed. In order to determine whether or not a cell is close to a hexagon, for example, image correlation processing with a hexagonal template image can be performed.
The cell orientation can be obtained based on, for example, a wire model created by thinning an image region.
The cell area is obtained by, for example, counting the number of pixels included in the unit area to obtain the unit area pixel number, counting the number of pixels in the cell region, and dividing the pixel number by the unit area pixel number. It is also possible to obtain the area by performing a normal integration operation.
The variation in cell area can be calculated by statistically processing the areas of a plurality of cells, for example.
The cell density may be calculated from the unit area of the observation visual field and the number of cells in the area.
The software used for these calculations is known and may be applied.
以下、実施例に基づいて本発明を更に詳細に説明する。なお、以下に説明する実施例は、本発明の代表的な実施例の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。 Hereinafter, the present invention will be described in more detail based on examples. In addition, the Example demonstrated below shows an example of the typical Example of this invention, and, thereby, the range of this invention is not interpreted narrowly.
＜重層上皮再構築体の作製＞
第一の培養工程：
ケラチノサイト（表皮角化細胞）を、細胞増殖用培地にて１×１０４ｃｅｌｌｓ／ｍＬの細胞懸濁液を調製し、１００ｍｍシャーレに１０ｍＬ添加する。その後、二酸化炭素濃度５％、３７℃、湿潤下にて、細胞が培養シャーレに対してサブコンフルエント若しくはコンフルエントになるまで７日間増殖させた。なお、培地は２日もしくは３日に一回交換した。
<Preparation of reconstructed stratified epithelium>
First culture step:
A cell suspension of 1 × 10 4 cells / mL of keratinocytes (epidermal keratinocytes) is prepared in a cell growth medium, and 10 mL is added to a 100 mm dish. Thereafter, cells were grown for 7 days under humid conditions at a carbon dioxide concentration of 5% at 37 ° C. until the cells became sub-confluent or confluent in the culture dish. The medium was changed once every 2 or 3 days.
第二の培養工程：
前記第一の培養工程で増殖した表皮角化細胞を酵素にてシャーレから剥がし、回収した。回収した細胞は細胞増殖用培地にて４×１０５ｃｅｌｌｓ／ｍＬに調製し、３次元培養用のセルカルチャーインサート（フィルター直径：１２ｍｍ、フィルター材質：ポリカーボネート）に５００μＬ添加し、インサートの外側には細胞増殖用培地をインサート内の液面と同じ高さになるまで添加した。細胞を播種した後、インサート底面に対して細胞がコンフルエントになるまで細胞増殖培地にて１日間培養させた。
Second culture step:
The epidermal keratinocytes grown in the first culture step were peeled off from the petri dish with an enzyme and collected. The collected cells are adjusted to 4 × 10 5 cells / mL in a cell growth medium, and 500 μL is added to a cell culture insert for three-dimensional culture (filter diameter: 12 mm, filter material: polycarbonate). The cell growth medium was added until the same level as the liquid level in the insert. After seeding the cells, the cells were cultured in a cell growth medium for 1 day until the cells became confluent with respect to the bottom surface of the insert.
第三の培養工程：
前記第二の培養工程で上皮細胞がコンフルエントになった後、培地を細胞分化用培地（Ｃａイオン濃度１．２ｍＭ）に変えた。上皮細胞の分化誘導を行うため、細胞分化用培地で１６時間浸漬培養した。
Third culture step:
After epithelial cells became confluent in the second culture step, the medium was changed to a cell differentiation medium (Ca ion concentration 1.2 mM). In order to induce differentiation of epithelial cells, the cells were immersed in a cell differentiation medium for 16 hours.
第四の培養工程：
前記第三の培養工程の培地を取り除き、上皮細胞の表面を気相（空気）に曝露し、細胞底面は細胞分化用培地に触れている状態で更に培養した。気相に曝露することで細胞が積層化し、角質層が形成された。その過程を図３に示す。
１２〜１４日後にヒトの表皮と同様の構造を持つ重層上皮再構築体を得た。
Fourth culture step:
The medium of the third culture step was removed, the surface of the epithelial cells was exposed to the gas phase (air), and further cultured with the cell bottom touching the cell differentiation medium. The cells were laminated by exposure to the gas phase, and a stratum corneum was formed. The process is shown in FIG.
After 12 to 14 days, a stratified epithelial reconstruct with the same structure as the human epidermis was obtained.
＜重層上皮再構築体のテープストリッピング＞
相対湿度９０％の条件下で、重層上皮再構築体を作製し、空気曝露１２日後にインサート底面のフィルターを繰り抜いた。次に、重層上皮再構築体の上面にテープを載せ、軽くひとなでした後にテープを剥がした。この重層上皮再構築体を反射式共焦点レーザー顕微鏡で観察した。
なお、テープには、ニチバン社製のセロテープ（登録商標）品番ＣＴ４０５ＡＰ−１８、幅１８ｍｍのものを用いた。
<Tape stripping of reconstructed stratified epithelium>
Under the condition of 90% relative humidity, a stratified epithelial reconstructed body was prepared, and the filter on the bottom surface of the insert was pulled out 12 days after air exposure. Next, a tape was placed on the upper surface of the stratified epithelial reconstructed body, and after lightly stroking, the tape was peeled off. This stratified epithelial reconstructed body was observed with a reflective confocal laser microscope.
The tape used was a cello tape (registered trademark) product number CT405AP-18 manufactured by Nichiban Co., Ltd., having a width of 18 mm.
＜結果＞
前記被験物質を適用しなかった重層上皮再構築体のテープストリッピング前の反射式共焦点レーザー顕微鏡像を図４に、テープストリッピング後の像を図５に示す。
テープストリッピングをすることで、図５に見られるように、初めて角質細胞の六角形状の形を確認することができた。
<Result>
FIG. 4 shows a reflection confocal laser microscope image before tape stripping of the stratified epithelial reconstructed body to which the test substance was not applied, and FIG. 5 shows an image after tape stripping.
By stripping the tape, the hexagonal shape of the keratinocytes could be confirmed for the first time as seen in FIG.
＜被験物質の評価＞
被験物質として、保湿剤であるグリセリンを滅菌水で１０質量％に調整したものを用いた。
相対湿度４０％の条件下で、インサートで培養したままの状態で、重層上皮再構築体の表面約１１３ｍｍ２（インサートのフィルターの底面積と同等）に被験物質の溶液を２００μＬ載せ、６０分静置後、被験物質の溶液を取り除いた。この操作を１日２回、計３日間実施した。
<Evaluation of test substance>
As a test substance, a humectant glycerin adjusted to 10% by mass with sterilized water was used.
200 μL of the test substance solution was placed on the surface of the stratified epithelial reconstructed body at approximately 113 mm 2 (equivalent to the bottom area of the filter of the insert) in a state where the relative humidity was 40% and cultured for 60 minutes. After placement, the test substance solution was removed. This operation was performed twice a day for a total of 3 days.
被験物質曝露後、重層上皮再構築体の付いたインサートの底面のフィルターを繰り抜いた。次に、重層上皮再構築体の上面にテープを載せ、軽くひとなでした後にテープを剥がした。この重層上皮再構築体を反射式共焦点レーザー顕微鏡で観察した。 After exposure to the test substance, the filter on the bottom surface of the insert with the stratified epithelial reconstructed body was pulled out. Next, a tape was placed on the upper surface of the stratified epithelial reconstructed body, and after lightly stroking, the tape was peeled off. This stratified epithelial reconstructed body was observed with a reflective confocal laser microscope.
前記被験物質の代わりに水を適用し、テープストリッピングをした重層上皮再構築体の共焦点レーザー顕微鏡像を図６に示す。前記被験物質を適用し、テープストリッピングをした重層上皮再構築体の像を図７に示す。
図６と図７を比較すると、水を適用した重層上皮再構築体では、角質細胞の六角形がほとんど確認できず、細胞の配向、配列等も乱れていたのに対し、前記被験物質を適用した重層上皮再構築体では、角質細胞のきれいな六角形と整然とした配向、配列等を確認できた。
以上のことから、重層上皮再構築体の上面を除去し、角質細胞の形、配向、配列等を観察することにより、被験物質の効果を評価できることが明らかとなった。
FIG. 6 shows a confocal laser microscope image of the stratified epithelial reconstructed body obtained by applying water instead of the test substance and tape stripping. FIG. 7 shows an image of the stratified epithelial reconstructed body to which the test substance is applied and subjected to tape stripping.
Comparing FIG. 6 and FIG. 7, in the stratified epithelial reconstructed body to which water was applied, the hexagonal shape of the keratinocytes could hardly be confirmed, and the cell orientation and arrangement were disturbed. In the stratified epithelial reconstructed body, clean hexagonal and orderly orientation and arrangement of keratinocytes could be confirmed.
From the above, it was revealed that the effect of the test substance can be evaluated by removing the upper surface of the stratified epithelial reconstructed body and observing the shape, orientation, arrangement, etc. of the corneocytes.
本発明によれば、ヒト等の動物の生体皮膚を用いずに、化粧料、皮膚外用剤、体内投与する薬剤等の皮膚に対する安全性試験、生理試験、スクリーニング等を視覚的に評価することができる。 According to the present invention, it is possible to visually evaluate safety tests, physiological tests, screenings, etc. on skin of cosmetics, external preparations for skin, drugs administered in the body, etc. without using living skin of animals such as humans. it can.
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